Electrochemical biosensor test strip, fabrication method thereof and electrochemical biosensor

ABSTRACT

Electrochemical biosensor test strip, fabrication method thereof and electrochemical biosensor are disclosed. The electrochemical biosensor test strip is fabricated by cutting a groove in a first insulation base in the breadth direction, forming two electrodes parallel to length direction on the first insulation base by sputtering using shadow mask, fixing a reaction material comprising an enzyme which reacts an analyte and generates current corresponding to the concentration of analyte across the two electrodes on the groove of the insulation base, and affixing a cover to the first insulation base. The groove of the first insulation base and the cover make a capillary at the position where the reaction material is fixed. The fabrication method can lower the cost for fabricating the test strip by forming thin electrodes.

CROSS REFERENCE TO RELATED APPLICATION

This application claims the priority of Korean patent application Ser.No. 1999-11810 filed on Apr. 6, 1999 and Korean patent application Ser.No. 1999-47573 filed on Oct. 29, 1999.

TECHNICAL FIELD

The present invention relates to an electrochemical biosensor test stripfor quantitative analysis of analytes of interest, a method forfabricating the same, and an electrochemical biosensor using the same.

BACKGROUND

In the medical field, electrochemical biosensors are extensively used toanalyze biomaterials, including blood. Of them, enzyme-utilizingelectrochemical biosensors are most predominant in hospital or clinicallaboratories because they are easy to apply and superior in measurementsensitivity, allowing the rapid acquisition of test results. Forelectrochemical biosensors, electrode methods have recently beenextensively applied. For example, in an electrode system established byscreen printing, the quantitative measurement of an analyte of interestcan be achieved by fixing a reagent comprising an enzyme onto theelectrodes, introducing a sample, and applying an electric potentialacross the electrodes.

An electrochemical biosensor using such an electrode method may bereferred to U.S. Pat. No. 5,120,420, which discloses an electrochemicalbiosensor test strip taking advantage of a capillary space for theintroduction of analytes, teaching the use of a spacer between aninsulating substrate and a cover to form the capillary space.

Another electrochemical biosensor test strip can be found in U.S. Pat.No. 5,437,999, in which a patterning technique, typically used in thePCB industry, is newly applied for the fabrication of an electrochemicalbiosensor, leading to an achievement of precisely defined electrodeareas. This electrochemical biosensor test strip is allegedly able toprecisely determine analyte concentrations on a very small sample size.

With reference to FIG. 1, there is an opposing electrode type of anelectrochemical biosensor test strip described in U.S. Pat. No.5,437,999, specified by a disassembled state in an exploded perspectiveview of FIG. 1A and by an assembled state in a perspective view of FIG.1B. Typically, these sensors perform an electrochemical measurement byapplying a potential difference across two or more electrodes which arein contact with a reagent and sample. As seen in the figure, theelectrochemical biosensor test strip comprises two electrodes: a workingelectrode on which reactions occur and a reference electrode whichserves as a standard potential.

There are two ways of arranging such working and reference electrodes.One is of an opposing electrode type just like that shown in FIG. 1A, inwhich a working electrode formed substrate is separated from a referenceelectrode by a spacer in a sandwich fashion. The other is of an adjacenttype in which a working and a reference electrode both are fabricated onthe same substrate side-by-side in a parallel fashion. U.S. Pat. No.5,437,999 also discloses an adjacent electrode electrochemicalbiosensor, adopting a spacer that separates an insulating substrate, onwhich the electrodes are fabricated, from another insulating substrate,which serves as a cover, forming a capillary space.

In detail referring to FIG. 1, a reference electrode-formed substrate,that is, a reference electrode element 10, is spatially separated from aworking electrode-formed substrate, that is a working electrode element20 by a spacer 16. Normally, the spacer 16 is affixed to the referenceelectrode element 10 during fabrication, but shown separate from thereference electrode element 10 in FIG. 1A. A cutout portion 13 in thespacer 16 is situated between the reference electrode element 10 and theworking element electrode 20, forming a capillary space 17. A firstcutout portion 22 in the working electrode element 20 exposes a workingelectrode area, which is exposed to the capillary space 17. When beingaffixed to the reference electrode element 10, a first cutout portion 13in the spacer 16 defines a reference electrode area 14, shown in phantomlines in FIG. 1, which is also exposed to the capillary space 17. Secondcutout portions 12 and 23 expose a reference electrode area 11 and aworking electrode area 21 respectively, serving as contact pads throughwhich an electrochemical biosensor test strip 30, a meter and a powersource are connected to one another.

In an assembled state as shown in FIG. 1B, the electrochemical biosensortest strip 30 has a first opening 32 at its one edge. Further, a ventport 24 in the working electrode element 20 may be incident to a ventport 15 in the reference electrode element 10 so as to provide a secondopening 32. In use, a sample containing an analyte may be introducedinto the capillary space 17 via either the opening 31 or 32. In eithercase, the sample is spontaneously drawn into the electrochemicalbiosensor test strip by capillary action. As a result, theelectrochemical biosensor test strip automatically controls the samplevolume measured without user intervention.

However, preexisting commercially available electrochemical biosensortest strips, including those described in the patent references supra,suffer from a serious problem as follows: because electrodes areplanarity fabricated on substrates and reagents, including enzymes, areimmobilized on the electrodes, liquid phases of the reagents arefeasible to flow down during the immobilization, so that they are verydifficult to immobilize in certain forms. This is highly problematic interms of the accuracy of detection or measurement because there is apossibility that the reagent immobilized on the electrodes might bedifferent from one to another every test strip. In addition, theelectrode area exposed to the capillary space is limitedly formed in theplanar substrates which the electrodes occupy. In fact, a narrowerelectrode area is restricted in detection accuracy.

U.S. Pat. No. 5,437,999 also describes methods for the fabrication ofelectrodes for electrochemical biosensor test strips, teaching atechnique of patterning an electrically conducting material affixed ontoan insulating substrate by use of photolithography and a technique ofscreen printing an electrically conducting material directly onto astandard printed circuit board substrate.

Photolithography, however, usually incurs high production cost. Inaddition, this technique finds difficulty in mass production because itis not highly successful in achieving fine patterns on a large area.

As for the screen printing, it requires a liquid phase of anelectrically conducting material. Although suitable as electricallyconducting materials for electrodes by virtue of their superiority indetection performance and chemical resistance, liquid phases of noblemetals, such gold, palladium, platinum and the like, are very expensive.Instead of these expensive noble metals, carbon is accordingly employedin practice. The electrode strip obtained by the screen printing ofcarbon is so significant uneven in its surface that its detectionperformance is low.

There is also suggested a method for fabricating an electrode for anelectrochemical biosensor test strip, in which a thick wire, obtained bydepositing palladium onto copper, is bonded on a substrate such asplastic film by heating. This method, however, suffers from adisadvantage in that it is difficult for the electrode strip to be of anarrow, thin shape owing to its procedural characteristics. As theelectric charges generated by the reaction between reagents and samplesare nearer to the electrodes, they are more probable to be captured anddetected by the electrodes. Hence, the bonding of a thick wire onto aplastic film brings about a decrease in the detection efficiency of theelectrochemical biosensor test strip. Further, detachment easily occursbetween the thick wire and the plastic film owing to a weak bondingstrength therebetween and the thick electrode requires high materialcost.

SUMMARY OF THE INVENTION

Therefore, it is an object of the present invention to provide anelectrochemical biosensor test strip which can firmly fix appropriatereagents in a certain pattern and secure a maximal effective area of anelectrode to detect charges, thereby enabling the precise quantitativedetermination of analytes of interest.

It is another object of the present invention to provide a method forfabricating such an electrochemical biosensor test strip, which iseconomically favorable as well as gives contribution to the precisedetection of analytes by forming an electrode of a uniform surface.

In accordance with an embodiment of the present invention, there isprovided an electrochemical biosensor test strip, comprising a firstinsulating substrate having a groove in a widthwise direction; a pair ofelectrodes parallel in a lengthwise direction on the first insulatingsubstrate; a reagent for reacting with an analyte of interest togenerate a current corresponding to the concentration of the analyte,the reagent being fixed in the groove of the first insulating substrate;and a second insulating substrate bonded onto the first insulatingsubstrate, the second insulating substrate forming a capillary space,along with the groove.

In accordance with another embodiment of the present invention, there isprovided a method for fabricating an electrochemical biosensor teststrip, comprising the steps of: forming a groove in a first insulatingsubstrate in a widthwise direction; sputtering a metal material onto thefirst insulating substrate with the aid of a shadow mask to form a pairof electrodes parallel in a lengthwise direction on the first insulatingsubstrate; fixing a reagent within the groove of the first insulatingsubstrate across a pair of the electrodes, the reagent reacting with ananalyte of interest to generate a current corresponding to theconcentration of the analyte; and bonding a second insulating substrateonto the first insulating substrate, the second insulating substrateforming a capillary space, along with the groove in which the reagent isfixed.

In accordance with a further embodiment of the present invention, thereis provided a method for fabricating an electrochemical biosensor teststrip, comprising the steps of: sputtering a metal material onto a firstinsulating substrate with the aid of a shadow mask to form a pair ofelectrodes parallel in a lengthwise direction on the first insulatingsubstrate; fixing a reagent on the first insulating substrate across apair of the electrodes, the reagent reacting with an analyte of interestto generate a current corresponding to the concentration of the analyte;and bonding a second insulating substrate having a groove in a widthwisedirection onto the first insulating substrate, the groove beingpositioned across the electrodes and forming a capillary space, alongwith the groove, at an area corresponding to the reagent fixed.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects, features and other advantages of thepresent invention will be more clearly understood from the followingdetailed description taken in conjunction with the accompanyingdrawings, in which:

FIG. 1 shows an opposing electrode type of a conventionalelectrochemical biosensor test strip, specified by a disassembled statein an exploded perspective view of FIG. 1A and by an assembled state ina perspective view of FIG. 1B;

FIG. 2 schematically shows a structure of an electrochemical biosensortest strip according to the present invention in perspective views;

FIG. 3 shows a process for fabricating a test strip in accordance with afirst embodiment of the present invention;

FIG. 4 is a schematic illustration of an process room in whichelectrodes of a test strip are fabricated by sputtering with the aid ofa shadow mask, in accordance with the present invention;

FIG. 5 shows a sputtering process with the aid of an adhesive-typeshadow mask in schematic cross sectional views; and

FIG. 6 shows a process for fabricating a test strip in accordance with asecond embodiment of the present invention.

DETAILED DESCRIPTION

The application of the preferred embodiments of the present invention isbest understood with reference to the accompanying drawings, whereinlike reference numerals are used for like and corresponding parts,respectively. The preferred embodiments are set forth to illustrate, butare not to be construed to limit the present invention.

With reference to FIG. 2, there is schematically shown a structure of anelectrochemical biosensor test strip according to the present inventionin perspective views. As seen, the electrochemical biosensor test stripof the present invention comprising an insulating substrate 41 or 42 onwhich a groove 45 or 46 is formed by embossing with a pressing or avacuum molding technique (FIG. 2A) or by engraving (FIG. 2B). Anelectrode 44 is installed on the insulating substrate 41 or 42. Thegroove 45 or 46, whether embossed or engraved, has a function of makingsure of the fixation of appropriate reagents (not shown) thereonto.

In such a structure of the electrochemical biosensor test stripaccording to the present invention, therefore, the reagents do not flowover the substrate 41 or 42 while being fixed onto the groove 45 or 46.In other words, the electrochemical biosensor test strip shown in FIG. 2allows reagents to be immobilized in a certain pattern, thereby makingthem constant enough to precisely detect or measure analytes ofinterest.

In addition, as shown in FIG. 2, the electrode installed in the teststrip according to the present invention has a three-dimensionalstructure, so that the electrode area exposed to a capillary space canbe further increased as much as an area corresponding to the groovedepth (deviant line). This indicates an increase in the electrode areacapable of capturing the charges generated by a reagent, resulting in animprovement in detection efficiency.

As illustrated above, the conventional techniques such as screenprinting methods and thick-wire bonding methods cannot establish such aprecise three-dimensional structure of an electrode in anelectrochemical biosensor test strip.

Below, a detail description will be given of a novel method which isable to establish such a precise three-dimensionally structuralelectrode in an electrochemical biosensor test strip, taking advantagesover the conventional methods.

With reference to FIG. 3, there is illustrated a method for fabricatingan electrochemical biosensor test strip in accordance with a firstembodiment of the present invention.

First, two metal electrode strips 52 and 54 are, in parallel, formed onan insulating substrate 50, one metal electrode strip offering a site ofoxidation as a working electrode 52, the other metal electrode stripserving as a corresponding reference electrode 54.

For use in the insulating substrate 50, any material is possible if theyare of an electrically insulating property, but in order to produce theelectrochemical biosensor test strip of the present invention on massproduction, preferable are those which possess flexibility large enoughto overcome roll processing as well as sufficient rigidity to berequired for supports. Suggested as such insulating substrate materialsare polymers, examples of which include polyester, polycarbonate,polystyrene, polyimide, polyvinyl chloride, polyethylene with preferenceto polyethylene terephthalate.

The formation of the electrode strips 52 and 54 on the insulatingsubstrate 50 is achieved by a sputtering technique with the aid of ashadow mask. In detail after a shadow mask in which an electrode stripcontour is patterned is arranged on the insulating substrate 50, atypical sputtering process is conducted, and removal of the shadow maskleaves the electrode strips 52 and 54 on the insulating substrate 50. Inthis regard, a pre-treatment, such as arc discharging or plasma etching,over the insulating substrate brings about an improvement in the bondingstrength between the insulating substrate and the electrode strips. Infact, when an electrode is formed of gold (Au) on an arc-treated plasticfilm, the bonding strength between the electrode and the insulatingsubstrate was found to be almost perfect (100%) as measured by a tapingtest.

Referring to FIG. 4, there is shown an process room in which a teststrip is formed by sputtering with the help of a shadow mask. In thisfigure, a target is designated as reference numeral 71, a plurality ofdot magnets as reference numeral 72, an iron plate as reference numeral73, an insulating substrate as reference numeral 74, a shadow mask asreference numeral 75, and areas in which electrodes are to be formed asreference numeral 76. Upon sputtering, the mask 75 and the substrate 74must be in close contact with each other. If there exists a gaptherebetween, however small it is, the material to be deposited, e.g.,gold, penetrates the gap, thereby resulting in a collapsed pattern. Inthe present invention, a plurality of dot magnets are employed to bringthe shadow mask into a close contact with the insulating substrate 74.In this regard, if the shadow mask 75 is thick, it cannot be affixed tothe magnets owing to its own mass and distortion. The experimental dataobtained by the present inventors show that a preferable thickness ofthe shadow mask 75 falls into the range of 0.1 to 0.3 mm.

In accordance with the present invention, the magnets are preferablyarranged in an inverse dot pattern. That is, the iron plate 73 is placedon the dot magnets 72. In this case, because the distortion of plasmahardly occurs, a great reduction can be brought about in the distancebetween the substrate 74 and the target 71, giving rise to a greatincrease in deposition efficiency.

Where plasma is generated, the process room is feasible to be heated tothe temperature at which commonly used plastic films are distorted. Inthis case, therefore, aluminum alloy, which shows high thermaltransmission properties and paramagnetic properties, such as SUS 430, isused as the shadow mask.

Suitable for use in electrodes are noble metals. Examples of the noblemetals include palladium, platinum, gold, silver and so on by virtue ofsuperior electrochemical properties in terms of stability on electrodesurface regions, electrochemical reproductivity, resistance tooxidation, etc. Particularly preferable is gold which enjoys advantagesof being relatively inexpensive, simple to process, superb inadhesiveness to plastic, and high in electrical conductivity. Althoughan electrode is formed of gold at as thin as 100 nm by sputtering, it issuitable as a disposable one because it has an electrically lowresistance and is mechanically firmly affixed to an insulating substratesuch as a plastic film. Alternatively, rather than such noble metalsonly, metal materials which are highly adhesive to insulatingsubstrates, such as plastics, and are inexpensive, may be used to form aprimary electrode on which the noble metal is thinly covered, for aneconomical reason.

Returning to FIG. 3B, a reagent 56 reactive to analytes is affixed witha suitable width across the two electrodes 52 and 54 on the insulatingsubstrate 50. The electrochemical biosensor test strip of the presentinvention can target a broad spectrum of analytes. Body materials, suchas whole blood, blood serum, urine, neurotransmitters and the like, aswell as fermented or naturally occurring materials can be detected ormeasured by the electrochemical biosensor test strip of the presentinvention. The reagent 56 can be coated on the electrode area of theinsulating substrate 50 with the aid of an automatic dispenser or by useof a screen printing, a roll coating, or a spin coating technique. Whenan electric potential is applied across the two electrodes after asample is provided, the reagent reacts with the sample in a reactiontime period to generate charges. Because these charges, which aregenerated through enzymatic reactions, relates to the concentration ofthe analyte of interest, the quantitative determination of the chargesprovides knowledge in regard to the concentration of the analyte.

Available as the reagent 56 are enzymes or redox mediators. A variety ofenzymes can be used in dependence on the analytes to be detected ormeasured. For example, when glucose is to be detected or analyzed,glucose oxidase may be used. Useful redox mediators may be exemplifiedby potassium ferricyanide and an imidazole osmium mediator which isdisclosed in U.S. Pat. No. 5,437,999. Besides enzymes and redoxmediators, the reagent 56 may further comprise buffers, hydrophilicmacromolecules, surfactants, and/or film-forming agents. During thereaction with a sample, a buffer in the reagent functions to keep a pHcondition constant. On the other hand, the hydrophilic macromoleculesare useful to fix other reagent components onto the electrode.Meanwhile, surfactants facilitate the introduction of samples into acapillary space, which will be explained later, by capillary action.Thus, the reagent for the detection or measurement of glucose maycomprise potassium ferricyanide, a potassium phosphate buffer,cellulose, hydroxyethyl cellulose, a Triton X-100 surfactant, sodiumsuccinate, and glucose oxidase in combination. A detailed preparationmethod of such reagents, and available enzymes and redox mediators canbe referred to U.S. Pat. No. 5,762,770.

With reference to FIG. 3C, an insulating plate 58 is fixed onto theelectrodes 52 and 54 and the insulating substrate by thermocompressionbonding or via a double-sided adhesive. FIG. 3D shows a profile of thestructure of FIG. 3C. As seen, the insulating plate 58 has a region tobe in contact with the electrodes 52 and 54 and the insulating substrate50 and a protruded region corresponding to the area onto which thereagent 56 is affixed. Suitable as a material for use in the insulatingplate 58 may be the same as the material for the insulating substrate50. Without being covered by the insulating plate 58, an upper part ofthe insulating substrate 50 remains bared. The electrodes 52 and 54,which are partially exposed at their upper parts, can serve as contactpads through which the electrochemical biosensor test strip, a meter anda power source are electrically connected to one another.

As shown in FIG. 3D, the protruded region of the insulating plate 58,along with the insulating substrate 50, forms a capillary space 64 whichtransverses the electrodes 52 and 54 in a widthwise direction. Thecapillary space needs not be completely as wide as, but may be wider ornarrower than the reagent 56. Likewise, the length of the capillaryspace also needs not be completely the same as, but may be greater orsmaller than the width of the insulating substrate 50. Only in order toreduce the error which occurs upon the introduction of a sample into thecapillary space, the length of the capillary space preferably agreeswith the width of the insulating substrate 50. The capillary space 64thus formed is where a sample such as blood is introduced. Thisintroduction is facilitated by a capillary action such that a precisedetermination can be done with even a small quantity of a sample.

Following is the principle of measuring the concentration of an analyteof interest, that is, a matter to be detected and/or analyzed, by use ofthe electrochemical biosensor test strip of the present invention. Whena glucose level in blood is assayed by use of a glucose oxidase withpotassium ferricyanide as a redox mediator, for instance, the glucose isoxidized while the ferricyanide is reduced into ferrocyanide, both beingcatalyzed by the glucose oxidase. After a predetermined period of time,when an electrical potential from a power source is applied across thetwo electrodes, a current is passed by the electron transfer attributedto the re-oxidation of the ferrocyanide. The electrical potentialapplied across the two electrodes from a power source is suitably notmore than 300 mV and preferably on the order of around 100 mV whentaking the properties of the mediator into account.

By applying a stored algorithm to the current meter, the current thusmeasured can be revealed as a dependent variable relative to theconcentration of the analyte in the sample. In another mathematicalmethod, by integrating the current measured in a current-time curveagainst a certain period of time, the total quantity of chargesgenerated during the time period can be obtained, which is directlyproportional to the concentration of the analyte. In brief theconcentration of an analyte in a sample can be quantitatively determinedby measuring the diffusing current which is generated by the enzymaticreaction-based electrical oxidation of a redox mediator.

Now, turning to FIG. 5, there are stepwise illustrated processes offabricating electrodes by sputtering with the aid of a adhesive-typeshadow mask.

A plastic film 80 is provided onto which a plastic film 84 as a shadowmask is attached via an adhesive layer 82, as shown in FIG. 5A. Theadhesive layer 82 is in an interim attachment state to the plastic film80, so they can be easily detached from each other.

Next, the plastic film 84 and the adhesive layer 82 are cut atpredetermined regions in the pattern of the electrodes to be formed,with the aid of a cutting plotter or an engraver, as shown in FIG. 5B.

Subsequently, the cut regions are taken off, followed by vacuumsputtering gold 88 wholly over the remaining structure to formelectrodes with the plastic film 84 being used as a shadow mask, asshown in FIG. 5C.

Finally, the remaining plastic film 84 and adhesive layer 82 are removedto bare the electrodes, as shown in FIG. 5D.

Like this case, an adhesive-type shadow mask allows patterns to beformed to the extent of the processing limit of a cutting plotter. Also,in contrast to typical iron shadow masks, such an adhesive-type shadowmask is flexible and attached to the film on which electrodes are to beformed, so that precise patterns can be established by sputteringwithout lateral diffusion.

Referring to FIG. 6, the method according to the present invention isapplied for the fabrication of an electrochemical biosensor test strip.

First, there is provided a plastic substrate 90 on which a structure ofan electrode strip is to be constructed, as shown in FIG. 6A.

Thereafter, a groove 92 is formed in a widthwise direction on theplastic substrate 92, as shown in FIG. 6B. In this regard, it ispreferred that both side banks 93 of the groove are slightly slantedlest gold electrodes, as will be deposited later, should be cut at theiredges. For the formation of the groove 92, a pressing or a vacuummolding method may be used to emboss the surface of the plasticsubstrate 90. Alternatively, the groove 92 can be formed by use of anengraver. The latter method is adapted to form the groove 92 of FIG. 6B.Since the matter for the plastic film 90 is usually wound around a roll,an engraver is more preferably used to groove the plastim film in lightof mass production. This procedure enables only two sheets of plasticfilm to be formed into an electrochemical biosensor test strip which hasa capillary space built-in, without additionally using a spacer as inU.S. Pat. No. 5,437,999.

Afterwards, electrode strips 94 and 95 are formed, as shown in FIG. 6C.For this, gold is vacuum sputtered onto the plastic substrate 90 withthe aid of a shadow mask, as previously mentioned. A reagent 98 iscoated within the groove 92 across the working electrode and thereference electrode and dried, as shown in FIG. 6D.

For the purpose of establishing such a three-dimensional structure of anelectrode strip as shown in FIG. 6C, the adoption of a planar shadowmask onto the grooved substrate makes a gap as high as the capillarytube between the mask and the substrate, through which gold from thetarget 71 penetrates, resulting in the formation of dull-definedpatterns. To avoid this problem, the following three techniques areemployed. First, the shadow mask is constructed so crookedly that itfits to the groove shape. By virtue of superb processability, SUS 430can be formed into such a three-dimensional structure of the shadowmask. Another solution is to control process parameters or the structureof the process room. The lower the pressure of the process room, thelonger the mean free path of the gold atoms sputtered. Thus, the atomsincident in the perpendicular direction onto the substrate become densein number. In other words, fewer atoms run in the lateral direction,resulting in the more precise definition of electrodes. In addition,lengthening the distance between the target 71 and the substrate 74makes a net flux of sputtered atoms perpendicular to the substrate 74.Where a five-inch circular target is employed, for example, almost nospread patterning is found if the distance from the substrate is over 7cm. The last measure the present invention takes to overcome the dulldefinition of electrode patterns is use of a collimator to block theatoms from running in a lateral direction. In contrast to ahoneystructure of collimators, usually used in semiconductor processes,the collimator used in the present invention is of a blind patternbecause it can restrict the running of atoms only in a lateraldirection.

Finally, an insulating plate 96 is bonded onto the plastic substrate 90in such a way that a major portion of the plastic substrate 90,including the groove 92, is covered with the insulating plate 96 whilethe other upper part remains uncovered, as shown in FIG. 6E. In result,the groove forms a capillary space, along with the insulating plate 96.Through the capillary space, a sample such as blood is introduced intothe electrochemical biosensor test strip. A profile of the finishedelectrochemical biosensor test strip of FIG. 6E is shown in FIG. 6F withan exaggerated illustration of the capillary space 99.

INDUSTRIAL APPLICABILITY

As described hereinbefore, the test strip of the present invention iscapable of precise quantitative determination of analytes of interest byvirtue of its firm fixation of appropriate reagents in a certain patternand of its possessing of a maximal effective area of an electrode todetect charges.

In addition, the method for fabricating such a test strip according tothe present invention thin electrode is economically favorable owing touse of the thin electrode films and gives contribution to the precisedetection of analytes by forming an electrode of a uniform surface fromgold, which is chemically stable.

The present invention has been described in an illustrative manner, andit is to be understood that the terminology used is intended to be inthe nature of description rather than of limitation. Many modificationsand variations of the present invention are possible in light of theabove teachings. Therefore, it is to be understood that within the scopeof the appended claims, the invention may be practiced otherwise than asspecifically described.

What is claimed is:
 1. An electrochemical biosensor test strip,comprising: a first insulating substrate having a groove in a widthwisedirection; a pair of electrodes parallel in a lengthwise directionacross the groove on the first insulating substrate; a reagent forreacting with an analyst of interest to generate a current correspondingto the concentration of the analyte, the reagent being fixed in thegroove of the fist insulating substrate; and a second insulatingsubstrate bonded into the first insulating substrate, the secondinsulating substrate forming a capillary space, along with the groove.2. The electrochemical biosensor test strip as set forth in claim 1,wherein the electrodes are formed of a noble metal selected from thegroup consisting of gold, silver, platinum and palladium.
 3. Theelectrochemical biosensor test stop as set forth in claim 1, wherein theelectrodes are formed of a double layer structure comprising a lowerlayer of a metal and an upper layer of a noble metal select from thegroup consisting of gold, silver, platinum and palladium.
 4. Theelectrochemical biosensor test strip as set forth in claim 1, whereinthe first insulating substrate is formed of a polymer selected from thegroup consisting of polyethylene terephthalate, polyester,polycarbonate, polystyrene, polyimide, polyvinyl chloride, andpolyethylene.
 5. A biosensor system, comprising: an electrochemicalbiosensor test stop of claim 1; and a detector for displaying an analyteconcentration in a sample, the detector being electrically connectedwith both the working electrode and the reference electrode, applying anelectric potential across the two electrodes, and measuring the currentgenerated as a result of the reaction between the reagent and thesample.
 6. A method for fabricating an electrochemical biosensor teststrip, comprising the steps of: forming a groove in a first insulatingsubstrate in a widthwise direction; sputtering a metal material onto thefirst insulating substrate with the aid of a shadow mask to form a pairof electrodes parallel in a longwise direction across the groove on thefirst insulating substrate; fixing a reagent within the groove of thefirst insulating substrate across a pair of the electrodes, the reagentreacting with an analyte of interest to generate a current correspondingto the concentration of the analyte; and bonding a second insulatingsubstrate onto the first insulating substrate, the second insulatingsubstrate forming a capillary space, along with the groove in which thereagent is fixed.
 7. The method as set forth in claim 6, wherein theelectrodes are formed of a noble metal selected from the groupconsisting of gold, silver, platinum and palladium.
 8. The method as setforth in claim 6, wherein the electrodes are formed of a double layerstructure comprising a lower layer of a metal and an upper layer of anoble metal selected from the group consisting of gold, silver, platinumand palladium.
 9. The method as set forth in claim 6, wherein the shadowmask is attached to the first insulating substrate by sue of a magnet.10. The method as set forth in claim 6, wherein the shadow mask isformed of an aluminum alloy which is excellent in thermal transmissionand magnetic properties.
 11. The method as set forth in claim 6, whereinthe shadow mask ranges, in thickness, from 0.1 to 0.3 mm.
 12. The methodas set forth in claim 6, wherein the sputtering step comprises: applyingan adhesive layer over the first insulating substrate to bond a maskfilm onto the first insulating substrate; cutting the mask film andadhesive in a desired pattern; removing the cut area from the mask filmand adhesive and depositing a metal element over the resultingstructure; and removing the remaining mask film and adhesive.
 13. Themethod as set forth in claim 6, wherein the fist insulating substrate isformed of a polymer selected from the group consisting of polyethyleneterephthalate, polyester, polycarbonate, polystyrene, polyimide,polyvinyl chloride, and polyethylene.
 14. The method as set forth inclaim 6, further comprising the step for conducting an arc dischargingor a plasma etching process over the first insulating substrate, priorto the sputtering step.
 15. The method as set forth in claim 6, whereinthe shadow mask has a three-dimensional structure suitable to fit to thegroove of the first insulating substrate.
 16. The method as set forth inclaim 6, wherein the sputtering step is conducted using a collimater.